The long-term objective of this proposal is to obtain a better understanding of the molecular mechanisms governing the first step in tumor cell dissemination, namely motility. To accomplish this we propose to define the structure-functional relationship and mechanism of action of an autocrine motility factor (AMF) and its receptor (AMFR/gp78). Phosphoglucose isomerase (PGI) is a ubiquitous housekeeping cytosolic enzyme that plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PGI acts as a potent mitogen/cytokine e.g. tumor autocrine motility factor (AMF), neuroleukin (NLK), and maturation factor (MF), sperm antigen-36 and myofibril-bound serine proteinase inhibitor and therefore represents a prototype for multifunction proteins i.e. moonlighting proteins. AMF/PGI contains the CXXC thioredoxin-box motif which resembles the CC and CXC motifs of chemokines and its signaling pathway involves cytoskeletal rearrangement and morphological alteration. We show here that AMF/PGI is a hypoxic inducible gene, which in turn up-regulates the expression of vascular endothelial growth factor (VEGF) and its receptor in endothelial cells and affects cellular oncogenic phenotype. AMFR, a 78 kDa seven-transmembrane glycoprotein is an orphan chemokine receptor, (gp78/AMFR/CXC2CR-1) that performs an additional function in the endoplasmic reticulum (ER) with an intrinsic RING finger-dependent ubiquitin protein ligase (E3) activity. Overexpression of AMF/AMFR has been found in a wide spectrum of malignancies, and regulates migration-dependent processes during invasion and metastasis in association with epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). In addition, we provided evidence that down-regulation of AMF/PGI expression results in loss of tumorigenicity leading to cell senescence. Thus, we hypothesize that there is cross-talk among glycolysis, hypoxia, cell growth and motility and that AMF/AMFR autocrine loop regulates, in part, tumor cell invasion and metastasis. Based on the above and the data outlined in the Progress Report we set the following Specific Aims for this renewal application: 1) To define the domains and mechanisms involved in AMF/AMFR regulated tumorigenicity and cell motility 2) To study the role of gp78/AMFR ubiquitin ligase in AMF ubiquitination and 3) To establish the role of AMF/AMFR in EMT'MET transitions. Central to targeting AMF/AMFR system and the relevance of this proposal to cancer biology will be the dissociation between cytokine and glycolytic activities as well as between the cell surface receptor and the intracellular ligase functions. This represents a major challenge of this proposal as it could bring about the development of specific small inhibitor molecules able to differentially inhibit a particular function without disturbing the other one and highlights its significance and novelty and would lead to a scientific evolution associated with cancer progression with the hope that these studies will result in better diagnosis, treatment and development of rational therapy. PUBLIC HEALTH RELEVANCE: The long-term objective of this proposal is to obtain a better understanding of the molecular mechanisms governing the first step in tumor cell dissemination, namely motility. To accomplish this we propose to define the structure-functional relationship and mechanism of action of an autocrine motility factor (AMF) and its receptor (AMFR/gp78). It is expected that these studies will lead to a better diagnosis, treatment and development of rational therapy.